The variability of the hepatitis B virus genome: statistical analysis and biological implications

A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.      

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Effects of short-term vibration and hypoxia during high-intensity cycling exercise on circulating levels of angiogenic regulators in humans.

This study aimed to investigate the biological response to hypoxia as a stimulus, as well as exercise- and vibration-induced shear stress, which is known to induce angiogenesis. Twelve male cyclists (27.8 +/- 5.4 yr) participated in this study. Each subject completed four cycle training sessions under normal conditions (NC) without vibration, NC with vibration, normobaric hypoxic conditions (HC) without vibration, and HC with vibration. Each session lasted 90 min, and sessions were held at weekly intervals in a randomized order. Five blood samples (pretraining and 0 h post-, 0.5 h post-, 1 h post-, and 4 h posttraining) were taken from each subject at each training session. Hypoxia was induced by a normobaric hypoxic chamber with an altitude of 2,500 m. The mechanical forces (cycling with or without vibration) were induced by a cycling ergometer. The parameters VEGF, endostatin, and matrix metalloproteinases (MMPs) were analyzed using the ELISA method. VEGF showed a significant increase immediately after the exercise only with exogenously induced vibrations, as calculated with separate ANOVA analysis. Endostatin increased after training under all conditions. Western blot analysis was performed for the determination of endostatin corresponding to the 22-kDa cleavage product of collagen XVIII. This demonstrated elevated protein content for endostatin at 0 h postexercise. MMP-2 increased in three of the four training conditions. The exception was NC with vibration. MMP-9 reached its maximum level at 4 h postexercise. In conclusion, the results support the contention that mechanical stimuli differentially influence factors involved in the induction of angiogenesis. These findings may contribute to a broader understanding of angiogenesis.     

Intense Resistance Exercise Induces Early and Transient Increases in Ryanodine Receptor 1 Phosphorylation in Human Skeletal Muscle

Background

While ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³) results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise.

Purpose

Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers.

Methods

7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques.

Results

While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01) post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05). pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01) in type I and II myofibers and in whole skeletal muscle.

Conclusion

Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and concomitantly pAMPKThr¹⁷² levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser²⁸⁴³ can mechanistically impact Ca²⁺handling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.     

Higher temperature reduces the effects of litter quality on decomposition by aquatic fungi

1. We investigated the effects of riparian plant diversity (species number and identity) and temperature on microbially mediated leaf decomposition by assessing fungal biodiversity, fungal reproduction and leaf mass loss. 2. Leaves of five riparian plant species were first immersed in a stream to allow microbial colonisation and were then exposed, alone or in all possible combinations, at 16 or 24 °C in laboratory microcosms. 3. Fungal biodiversity was reduced by temperature but was not affected by litter diversity. Temperature altered fungal community composition with species of warmer climate, such as Lunulospora curvula, becoming dominant. 4. Fungal reproduction was affected by litter diversity, but not by temperature. Fungal reproduction in leaf mixtures did not differ or was lower than that expected from the weighted sum of fungal sporulation on individual leaf species. At the higher temperature, the negative effect of litter diversity on fungal reproduction decreased with the number of leaf species. 5. Leaf mass loss was affected by the identity of leaf mixtures (i.e. litter quality), but not by leaf species number. This was mainly explained by the negative correlation between leaf decomposition and initial lignin concentration of leaves. 6. At 24 °C, the negative effects of lignin on microbially mediated leaf decomposition diminished, suggesting that higher temperatures may weaken the effects of litter quality on plant litter decomposition in streams. 7. The reduction in the negative effects of lignin at the higher temperature resulted in an increased microbially mediated litter decomposition, which may favour invertebrate‐mediated litter decomposition leading to a depletion of litter stocks in streams.     

Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.